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RESEARCH ARTICLE

Marker-assisted introgression of opaque2 allele for rapid conversion of elite hybrids into quality

protein maize

Firoz Hossain*, Vignesh Muthusamy, Neha Pandey, Ashish K. Vishwakarma, Aanchal Baveja,

Rajkumar U. Zunjare, Nepolean Thirunavukkarasu, Supradip Saha, Kanchikeri M. Manjaiah,

Boddupalli M. Prasanna# and Hari S. Gupta

ICAR-Indian Agricultural Research Institute, New Delhi-110012, India;

#Present address: International Maize and Wheat Improvement Centre (CIMMYT), Nairobi, Kenya;

*Corresponding author: fh_gpb@yahoo.com

Running title:

MAS for development of QPM hybrids

Keywords. Biofortification; essential amino acids; opaque2; QPM; marker-assisted selection

Abstract

Maize is a valuable source of food and feed worldwide. Maize endosperm protein is, however

nutritionally poor due to reduced levels of two essential amino acids, lysine and tryptophan. In this

study, recessive opaque2 (o2) allele that confers enhanced endosperm lysine and tryptophan, was

introgressed using marker-assisted backcross breeding into three normal inbred lines (HKI323,

HKI1105 and HKI1128). These are the parental lines of three popular medium-maturing single cross

hybrids (HM4, HM8 and HM9) in India. Gene-based simple sequence repeat (SSR) markers (umc1066

and phi057) were successfully deployed for introgression of o2 allele. Background selection using

genome-based SSRs helped in recovering >96% of recurrent parent genome. The newly developed

Quality Protein Maize (QPM) inbreds showed modified kernels (25-50% opaqueness) coupled with

high degree of phenotypic resemblance to the respective recipient lines, including grain yield. In

addition, endosperm pprotein quality showed increased lysine and tryptophan in the inbreds to the

range of 52-95% and 47-118%, respectively. The reconstituted QPM hybrids recorded significant

enhancement of endosperm lysine (48-74%) and tryptophan (55-100%) in the endosperm. The QPM

hybrids exhibited high phenotypic similarity with the original hybrids for morphological and yield

contributing traits along with responses to some major diseases like turcicum leaf blight and maydis

leaf blight. The grain yield of QPM hybrids was at par to their original versions under multi-location

testing. These elite, high yielding QPM hybrids with improved protein quality have been identified for

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release by All India Coordinated Research Project on Maize, and hold significant promise for

improving nutritional security.

Introduction

Protein-energy malnutrition (PEM) has emerged as a major health problem especially in the

developing world (Temba et al. 2016). Maize provides significant amount of total calorie to the human

populations worldwide. Energy requirement to the tune of 62% in Mesoamerica, 43% in Eastern and

Southern Africa, 22% in West and Central Africa, and 28% in the Andean region, comes from maize

(Shiferaw et al. 2011). It is also a preferred choice as a food in many of the tribal belts, and especially

in the North Eastern states. A major portion (60-70%) of maize grains produced worldwide is used for

animal consumption. Maize thus serves as an important source of plant protein and total calorie both

directly and indirectly. Maize endosperm protein is, however known to be poor in nutritional value

due to low amount of essential amino acids, lysine (2.0% in protein) and tryptophan (0.4% in protein),

and their concentration is nearly half of the level recommended for human nutrition (Mertz et al. 1964;

Prasanna et al. 2001). Since, humans and monogastric animals like poultry cannot synthesize these

amino acids in their body, healthy diets therefore must include the alternate sources of lysine and

tryptophan (Bjarnason and Vasal 1992; Gupta et al. 2013). Among various strategies, biofortification

turns out to be the most sustainable and cost-effective solution to provide micronutrients in natural

form (Bouis et al. 2011, Gupta et al. 2015).

The recessive opaque2 (o2) mutant has been successfully utilized in the breeding programme

for enhancement of protein quality (Vasal et al. 1980). Initially maize cultivars with o2 mutation was

not preferred by farmers and consumers, due to soft and opaque endosperm, increased susceptibility to

insect-pest and diseases, and breakage of grains during mechanical processing (Bjarnason and Vasal

1992). Later, endosperm modifier genes that confer hard endosperm in the o2 background were

introgressed at CIMMYT, Mexico (Villegas et al. 1992) and University of Natal, South Africa

(Geevers and Lake 1992). This eventually led to the development of nutritionally enriched hard

endosperm maize, popularly phrased as ‘Quality Protein Maize’ (Vasal et al. 1980).

Globally, a large number of normal maize hybrids have been released and commercialized. In

contrast, the germplasm base of QPM is quite narrow, and substantially lesser number of genetically

diverse QPM hybrids are available. In India, only a dozen QPM hybrids has been released, compared

to more than hundred non-QPM/normal maize hybrids (Yadav et al. 2015). It is therefore necessary to

develop diverse QPM varieties across different maturity groups and agro-ecologies. The conventional

approach takes 10-15 years or more to implement this program. Conversion of elite normal maize

hybrids into QPM requires significantly lesser time primarily due to tested -combining ability, -

heterosis and -adaptability of the already released hybrid (Prasanna et al. 2010). Introgression of a

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recessive allele through conventional backcross breeding involves 6-7 generations of backcrossing.

This time can be significantly reduced to two backcrosses by molecular marker-assisted backcross

breeding (MABB) (Babu et al. 2005; Muthusamy et al. 2014). We report here rapid conversion of

three popular medium-maturity single-cross hybrids released in India, HM4, HM8 and HM9, to QPM

using MABB.

Materials and methods

Plant materials

The genetic materials comprised of three elite normal/non-QPM maize inbreds, HKI323, HKI1105

and HKI1128 having low level of lysine (1.76-2.08% in protein) and tryptophan (0.35-0.52% in

protein) (Table 1). These are the parents of three commercial medium-maturing single-cross maize

hybrids in India [HM4 (HKI1105 × HKI323), HM8 (HKI1105 × HKI161) and HM9 (HKI1105 ×

HKI1128)]. HKI161 possesses o2 allele and is a QPM inbred. HM4 is adapted to North Western Plain

Zone (NWPZ), while HM8 and HM9 are for Peninsular Zone (PZ) and North Eastern Plain Zone

(NEPZ) of India (Kaul et al.2009). CML161 [CIMMYT QPM inbred], HKI161 and HKI193-1 [QPM

inbreds selected from CML161 and CML193, respectively at Uchani Centre, Chaudhary Charan Singh

Haryana Agricultural University (CCSHAU), India] with high levels of endosperm lysine (3.32-3.80%

in protein) and tryptophan (0.74-0.85% in protein), and desirable endosperm modification served as

the donors for introgression of o2 allele.

Target allele for introgression

Two SSRs, umc1066 and phi057 are present in first- and sixth-exon, respectively, of O2 gene present

on chromosome 7 (Yang et al. 2004). The primer sequences are: umc1066 = Forward (F): 5′-

ATGGAGCACGTCATCTCAATGG-3′; Reverse (R): 5′-AGCAGCAGCAACGTCTATGACACT-3′

and phi057 = F: 5′-CTCATCAGTGCCGTCGTCCAT-3’ and R: 5′-

CAGTCGCAAGAAACCGTTGCC-3′. Polymorphic SSRs between respective recurrent- and donor-

parents were used for marker-assisted selection (MAS) of the o2 allele.

DNA isolation and polymerase chain reaction

Genomic DNA was isolated from young seedlings using the standard CTAB procedure (Murray and

Thompson 1980). Polymerase chain reaction (PCR) reaction (Bio-Rad, California, USA) was carried

out in 10 μl of a reaction mixture containing 2 μl of 20 ng/μl genomic DNA as the template, 2 mM

MgCl2, 1 mM dNTPs, 2 μM of the primer pair (F and R), and 1.5 U Taq Polymerase (GeNei, Mumbai,

India). ‘Touch-down’ procedure standardised at Maize Genetics Unit, ICAR-Indian Agricultural

Research Institute (IARI), New Delhi, was used for PCR amplification (Pandey et al. 2015). The

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resulting PCR amplicons were resolved in 4% agarose gel for 4 hours. The resolved amplified

products were visualized using a gel documentation system (AlphaInnotech, California, USA).

MABB strategy

For MABB (Fig. 1), recurrent parents (as females) and donors (as males) were crossed in 2009 rainy

season (July to October) at IARI Experimental Farm, New Delhi. The F1s were raised at the Maize

Winter Nursery Centre (off-season nursery), Hyderabad, India, during 2009-10 winter season

(December to April). Heterozygosity of the F1s was tested using the o2-specific marker, and the true

F1s were backcrossed as male parents to their respective recurrent parents. BC1F1 progenies were

grown at Delhi during the rainy season in 2010, and foreground selection was carried out using the o2-

gene-specific marker(s). Desirable plants with high recovery of the recurrent parent genome (RPG)

and maximum morphological similarity to recurrent parents were further backcrossed to raise the

BC2F1 population at Hyderabad during 2010-11 winter season. The selected plants with high RPG and

phenotypic similarity to their recurrent parents were selfed. BC2F2 populations were raised at Delhi

during 2011 rainy season, and selected plants were selfed to advance progenies. Backcross progenies

of HKI323 × HKI161and HKI1105 × CML161 were generated as per the procedure as above.

However, for HKI1128 × HKI193-1, BC1F1 seeds could not be generated during winter of 2009-10

owing to the non-synchrony of flowering; hence, fresh crosses were generated and F1s were planted at

Delhi during 2010 rainy season, and all backcross progenies were eventually raised one generation

later compared to other two inbreds.

(a) Marker-assisted foreground selection:

Foreground selection was employed in BC1F1, BC2F1, and BC2F2 generations using the marker

specific to o2 allele. SSRs, umc1066 and phi057 were used for selection of foreground positive plants.

Heterozygous plants (O2/o2) were selected in the BC1F1 and BC2F1, and homozygotes (o2/o2) were

selected in BC2F2. Chi-square (χ2) test was performed for testing the goodness of fit of the observed

segregation pattern at the o2 locus in each of the generations.

(b) Marker-assisted background selection:

A set of >350 genome-wide SSRs distributed throughout the maize genome was used for identifying

polymorphic markers between the respective recurrent- and donor- parents. The sequences of the SSR

primers were adapted from the maize genome database (www.maizegdb.org) and custom-synthesized

(Sigma Tech., USA). These polymorphic SSR markers were employed in each of the BC1F1 and

BC2F1 generations of the three crosses to recover the RPG. The final recovery of RPG, across genetic

backgrounds, was determined in the BC2F4 generation.

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Endosperm modification

Selfed seeds (BC2F3 onwards) from homozygous plants (o2/o2) were selected and analysed for the

degree of opaqueness using a standard light box (Vasal et al. 1980). For analysis of endosperm

modification, the back-lit kernels were rated on a scale of 0 to 100, with each number indicating %

opaqueness. For instance, ‘100’ stands for 100% opaque while ‘0’ for 100% vitreous/hard (Hossain et

al. 2008). Grains with minimal degree of opaqueness were selected and used for advancement of

homozygous progenies (o2/o2).

Phenotypic characterization of MAS-derived inbreds

The MAS-derived inbreds along with the original inbreds were evaluated at Delhi, during 2013 and

2014 rainy season in two replications each having two-rows/entry. Inbreds were characterized for 31

morphological characters (PPVFRA 2007) and 12 grain yield-related traits. Plants were raised in 3m

length row with a plant to plant distance of 20 cm, and row to row distance of 75 cm. Standard

agronomic practices like application of 10-15 tonnes of farm yard manure (FYM), 150: 80: 60 kg of

N: P: K, and 20-25 kg ZnSO4 per ha in soil, and 6-8 irrigations depending upon the requirement were

given, to raise the crop.

Evaluation of MAS-derived hybrids

Since the hybrids targeted for improvement are adaptable for cultivation in rainy season, crosses were

generated in winter season at off season nursery, and reconstituted hybrids were evaluated in the

following rainy season. In rainy season, seeds of selected MAS-derived QPM inbreds were increased,

and progenies (BC2F5 for HKI-323Q and HKI-1105Q, and BC2F4 for HKI-1128Q) were crossed to

reconstitute the QPM version of original hybrid at Hyderabad, during 2012-13 winter season. HKI161

being a QPM inbred, was directly used as a parent for generating QPM version of HM8. The crosses

along with original hybrids were evaluated in two replications each having two-rows/entry at Delhi,

during 2013 rainy season. Standard agronomic practices used for raising inbreds were also followed to

raise the hybrids. The hybrids were characterized for 31 morphological characters (PPVFRA 2007)

and 12 grain yield-related traits.

Estimation of lysine and tryptophan in endosperm protein

Inbred trials conducted for assessing the grain yield and component traits at Delhi during 2013 and

2014, were used for estimation of lysine and tryptophan in endosperm protein. For hybrids, the yield

trial conducted in Delhi during 2013 was used for quality analyses. In addition, a separate hybrid trial

was constituted at Delhi in 2014 only for quality analyses. Two to three plants in each entry of the

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trials were self-pollinated, and selfed grains were used for estimation of grain quality. Total

endosperm protein was estimated using the micro-Kjeldahl procedure (AOAC 1965). The

concentration of lysine and tryptophan in endosperm flour was measured using UPLC (Dionex

Ultimate 3000, Thermo Scientific) at Maize Genetics Unit, IARI, New Delhi (Sarika et al. 2016).

Degermed endosperm flour per sample with three replications was used. Acid hydrolysis was done for

lysine, while for tryptophan alkaline hydrolysis was performed. Samples were eluted through

AcclaimTM 120 C18 column (5µm, 120 Å, 4.6 × 150mm, Thermo Scientific) and detected with RS

photodiode array detector (PDA) with absorbance at 265 and 280nm wavelength respectively.

Concentration of amino acids in each sample was estimated by standard regression using external

standards (AAS-18, Sigma Aldrich, USA).

Evaluation of MAS-derived hybrids in multi-location-based national trials

The MAS-derived experimental crosses evaluated at Delhi during 2013 rainy season, were further

nominated as ‘essentially derived variety’ (EDV) and tested under the AICRP-Maize, coordinated by

the ICAR-Indian Institute of Maize Research (IIMR), New Delhi. Under this system, each entry was

coded and the trial for each of the hybrids was undertaken at 3-12 designated locations of their

respective zone of adaptation. The entries were evaluated in complete randomized block design (three

replications and having four rows/entry/replications) for the two consecutive years (2014 and 2015

rainy seasons) (Annual Progress Report, Kharif Maize 2015, 2016). Various agronomic traits

including grain yield were recorded. Hybrids were also evaluated for their responses to diseases like,

maydis leaf blight, turcicum leaf blight, banded leaf and sheath blight, polysora rust, common rust,

charcoal rot, fusarium stalk rot, sorghum downy mildew, Rajasthan downy mildew and bacterial stalk

rot.

Results

Marker polymorphism

The umc1066 was polymorphic between recurrent parents (HKI323 and HKI1128) and donor QPM

inbreds (HKI161 and HKI193-1), respectively. For HKI1105 and CML161, phi057 was used as a

polymorphic. 351-463 genome-based SSR markers distributed among 10 chromosomes were screened

between the recipient- and donor-parents and 30-42% was polymorphic across three crosses (Table 2).

Marker-assisted introgression of o2 allele

(a) BC1F1 generation:

Foreground selection in BC1F1 resulted in the identification of 21 heterozygous plants in HKI323 ×

HKI161, 32 in HKI1105 × CML161 and 50 in HKI1128 × HKI193-1 populations (Table 3).

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Segregation of o2 allele in all the three populations deviated from the expected Mendelian ratio of 1:1

(Table 3), while recovery of RPG varied from 63.26 to 91.9% (Table 4). Two plants each in HKI323-

(73.6 and 74.3% RPG), HKI1105- (76.3 and 77.0% RPG) and HKI1128- (85.5 and 90.0% RPG) -

based populations were selected for further advancement (Table 4).

(b) BC2F1generation:

A total of 53 heterozygous plants (O2/o2) were identified in HKI323 × HKI161, while 57 in HKI1105

× CML161, and 68 in HKI1128 × HKI193-1 (Table 3). Significant segregation distortion of o2 allele

was observed in the first two crosses, while in third (HKI1128 × HKI193-1) it was 1:1 (Table 3).

Background selection in the heterozygous plants using polymorphic SSRs led to the recovery of 84.9-

93.9% RPG in HKI323 × HKI161, 82.1-93.8% in HKI1105 × CML161, and 89.6-94.0% in HKI1128 ×

HKI193-1. Two plants each in HKI323- (93.2 and 93.9% RPG) and HKI1128- (93.3 and 94.0% RPG)

were advanced, while three in HKI1105- (82.1%, 910% and 91.1% RPG) (Table 4).

(c) BC2F2 generation:

Foreground selection identified 58 homozygous plants (o2/o2) in HKI323 × HKI161, while the same

was 20 and 60 in HKI1105 × CML161 and HKI1128 × HKI193-1, respectively (Table 3). All the three

crosses deviated from the expected segregation pattern of 1:2:1 (Table 3). Homozygous plants (o2/o2)

with similarity to their respective recurrent parents were selected for advancement.

(d) BC2F4 generation:

The highest recovery of RPG observed was 98.0% in HKI323 × HKI161, 96.6% in HKI1105 ×

CML161 and 98.3% in HKI1128 × HKI193-1 (Table 4). Based on higher recovery of RPG, phenotypic

similarity to the recurrent parent and desirable degree of grain modification, HKI323-44-68-16,

HKI1105-22-99-3 and HKI1128-48-1-14 were finally selected and used for advancement.

Evaluation of introgressed inbreds for grain quality and yield attributes

(a) Endosperm lysine and tryptophan in MAS-derived inbreds:

Across years, 58.5-70.0% increase in lysine, and 46.0-96.0% enhancement in tryptophan was observed

in the selected progenies over their respective recurrent parents (Table 5). The concentration of protein

in endosperm remained almost same in both original- and introgressed inbreds. However, the protein

quality was significantly improved in MAS-derived inbreds (Table 5).

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(b) Endosperm modification in introgressed progenies:

For HKI323-based progenies, degree of opaqueness varied from 25-75%, while for HKI1105 and

HKI112, it was 25-50% and 50-100%, respectively. However, the MAS-derived inbred that was

selected for generating the hybrid combinations possessed 25% opaqueness for HKI323-44-68-16, and

50% for each of HKI1105-22-99-3 and HKI1128-HKI1128-48-1-14 (Table 4).

(c) Morphological characteristics of MAS-derived inbreds:

The MAS-derived inbreds showed high degree of resemblance with their respective recurrent parents

(Table S1). However, the introgressed inbreds differed with their original inbreds for very few

characters. For example, anthocyanin colouration in brace root and base of glume is present in

HKI1105, while absent in QPM of HKI1105 (HKI1105-22-99-3). The MAS-derived inbreds however,

possessed similar grain yield as achieved in original inbreds (Table 6).

Evaluation of reconstituted hybrids for grain quality-, yield- attributes and responses to diseases

Endosperm lysine and tryptophan in MAS-derived hybrids:

MAS-derived QPM version of HM4 (HKI1105-22-99-3 × HKI323-44-68-16), HM8 (HKI1105-22-99-

3 × HKI161) and HM9 (HKI1105-22-99-3 × HKI1128-48-1-14), were designated as HM4-Q, HM8-Q

and HM9-Q, respectively. The concentration of lysine and tryptophan in endosperm of reconstituted

hybrids also recorded significant improvement over their respective original hybrids. Based on both

the years, 50-78% enhancement in lysine was recorded, while tryptophan showed 51-100% increase

across hybrids (Table 7). Protein content remained almost same in both MAS-derived and original

hybrids, but the protein quality showed significant enhancement. The increase in lysine in protein was

in the range of 49.5 to 77.0%, while, the same for tryptophan in protein was 61.0 to 97.5% (Table 7).

(a) Evaluation of MAS-derived hybrids for morphological characteristics:

The reconstituted hybrids resembled their respective original hybrids with high degree of similarity

except a few (Table S2). The selfed F2 seeds of HM4-Q, HM8-Q and HM9-Q showed desirable degree

of endosperm modifications. Grain yield and other contributing traits were also similar among the

original and MAS-derived hybrids (Table 8).

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The data generated during 2014 and 2015 under the multi-location trials of AICRP-Maize,

clearly suggested that QPM version of reconstituted hybrids showed high degree of resemblance to

their original hybrids for grain yield and yield traits (Table S3). The flowering behaviour and maturity

of the reconstituted hybrids were similar to the original hybrids as well (Table S3).

Further, the reconstituted- and original- hybrids also showed high degree of resemblance for

resistance to important diseases of maize in the country (Table S4). For example, HM4 during 2014

had the disease score of 3.0 against maydis leaf blight, while the same was 2.4 for HM4-Q. HM8 and

HM9 recorded score of 3.0 and 2.8, while their QPM versions had 2.7 and 2.3, respectively. Similarly,

all the hybrids recorded moderate resistance against turcicum leaf blight during 2015 (Table S4).

Discussion

MABB has been employed to introgress low phytic acid (lpa) (Naidoo et al. 2012), o2 (Gupta et al.

2013) and crtRB1 (Muthusamy et al. 2014) alleles to improve nutritional quality traits in maize. In the

present investigation, distinct marker polymorphism between the respective recurrent- and donor-

parents facilitated the introgression of recessiveo2 allele into the elite inbreds. Since we used o2-

specific SSRs (located within the target gene), segregants could be selected with 100% efficacy (Babu

et al. 2005). We also found severe segregation distortion (SD) for the o2 as reported by Jompuk et al.

(2011). The reason for the SD could be the presence of many segregation distortion regions (SDRs)

throughout the maize genome (Lu et al. 2002). Frequent occurrence of SD thus necessitates generation

of large populations for obtaining sufficient numbers of foreground-positive plants (Muthusamy et al.

2014).

Introgression of o2 allele resulted in significant increase in protein quality by enhancing the

concentration of lysine and tryptophan in the endosperm of both MAS-derived inbreds and hybrids.

Lysine in protein recorded 48-95% enhancement, while tryptophan in protein showed 47-118%

increase across inbreds/hybrids over their respective genotypes. The mechanism of enhancement of

lysine and tryptophan in o2 genotypes are of diverse type. The enhancement of nutritional quality in

o2 mutant is mainly due to (i) reduction of lysine deficient zein proteins followed by enhanced

synthesis of lysine-rich non-zein proteins (Habben et al. 1993), (ii) reduced transcription of lysine

catabolizing enzyme, lysine keto-reductase, (Kemper et al. 1999), and (iii) enhanced synthesis of

various lysine-rich proteins and enzymes (Jia et al. 2013).

Though introgression of o2 allele alone has a major effect on accumulation of lysine and

tryptophan in higher concentrations, the levels of the same varied substantially across genetic

background. For example, lysine ranged from 0.277 to 0.373%, while tryptophan varied from 0.067 to

0.082% across three inbreds. The variation is due to amino acid modifier loci that influence the

accumulation lysine and tryptophan in o2genetic background (Pandey et al. 2015). The levels of lysine

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and tryptophan in MAS-derived hybrids also showed large variation, and interactions of amino acid

modifiers contributed by both the parents possibly determined the final level of the targeted amino

acids. Further, the lysine and tryptophan levels were lesser in HKI323-44-68-16 and HKI1105-22-99-

3, compared to their respective donor parents (HKI161 and HKI193-1, respectively). It is possibly due

to the fact that favourable modifier loci present in the QPM donor was lost owing to repeated

backcrossing to recurrent parent. QTLs for these modifier loci in QPM background have been

identified recently, and can be used along with o2 allele in future breeding programme (Babu et al.

2015).

SSRs covering all 10 chromosomes were used for recovering the major proportion of the RPG

within two backcross generations. The highest RPG recovery among the selected progenies was

96.58% in HKI1105 × CML161, while it was 97.97% and 98.35% in HKI323 × HKI161 and HKI1128

× HKI193-1, respectively. To achieve comparable results, conventional breeding would take five

backcrosses since o2 is recessive in nature. In conventional method, BC5F3 progeny would be crossed

with its other parent to reconstitute the hybrid. Thus, it would require 14 seasons from crossing the

recipient and donor to the evaluation of hybrids. In contrast, MABB approach used here took 8-9

seasons since BC2F4 (HKI323- and HKI1105-based)/ BC2F5 (HKI1128-based) progenies were crossed

to reconstitute the hybrids. The MABB approach thus clearly saved time of raising 5-6 additional

seasons, and therefore accelerated the pace of breeding (Gupta et al. 2013; Muthusamy et al. 2014).

Phenotypic features such as plant-, ear- and grain- characteristics used in combination with

MAS helped to recover the RPG even rapidly (Manna et al. 2005; Muthusamy et al. 2014). Though,

introgressed inbreds and reconstituted hybrids resembled their respective recurrent parents or original

hybrids for majority of characters, they also differed for a few characters. In fact, morphological

characteristics that show sharp contrast are highly useful for registration of genotypes (Gunjaca et al.

2008). Besides they also act as morphological marker to unambiguously differentiate the QPM-

versions from the original inbreds during seed production and certification. The contrasting features

are possibly due to the effects of minor proportion of donor genome (2.03-4.89%) present in the

introgressed progenies (Choudhary et al. 2014).

The selected MAS-derived o2 inbreds across three genetic backgrounds showed desirable

degree (25-50% opaqueness) of endosperm modification. CML161, HKI161 and HKI193-1 are the

popular QPM inbreds, and possess hard endosperm (50% opaqueness) due to the presence of

favourable endosperm modifier loci (Paez 1973; Pandey et al. 2015). Though no marker system was

used for the selection of endosperm modifiers in the backcross generations, the screening of kernels

from o2/o2 plants on light box could successfully select the desirable progenies that possessed higher

modification (Gupta et al. 2013).

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The grain yield potential of the reconstituted hybrids was at par with the original hybrids

across multiple locations. Selection of SSR alleles specific to recurrent parents indirectly led to the

selection of unknown loci associated with yield, agronomic traits and heterosis (Gupta et al. 2013;

Muthusamy et al. 2014). Further, the similar response recorded in reconstituted- and original- hybrids

against various diseases is also due to similar genomic constitution achieved through high recovery of

RPG. Here also, the loci involved in responses to various diseases were not selected in the backcross

progenies, but the background selection helped in retaining those unknown loci. However, in some

cases the responses in QPM versions were slightly different from the original hybrids. These minor

changes in response to diseases are due to the presence of minor fragments of the donor parent

genome. QPM versions of HM4, HM8 and HM9 have now been identified for release as the promising

hybrids for their respective zones under the AICRP-Maize during 2017. The newly developed QPM

hybrids with better protein quality, high grain yield and diverse adaptation offer promise in reducing

nutritional insecurity.

Acknowledgements

Financial support from the Department of Biotechnology, Government of India, through the project

entitled‘Rapid conversion of normal maize inbreds to quality protein maize and further enhancement

of limiting amino acids in elite inbreds through marker-assisted selection’

(BT/PR11708/AGR/02/649/2008) is gratefully acknowledged. The authors are thankful to Dr. Sain

Dass, former Director, ICAR-IIMR, New Delhi for his valuable suggestions. We also thank ICAR-

IIMR, New Delhi; CCSHAU, Uchani; and CIMMYT, Mexico, for sharing the inbred lines used in this

study. The support of AICRP-Maize in conducting the multi-location trials is duly acknowledged.

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Figure captions:

Fig. 1: Marker-assisted backcross breeding scheme adopted for conversion of normal maize

hybrids into QPM versions.

15

Table 1: Details of the recurrent and donor parents used in the study

No. Name* Derived from

Recurrent parents

1. HKI323 CIMMYT Pool 28

2. HKI1105 Cargil 633

3. HKI1128 Hybrid from a farmer’s field

Donor parents

1. HKI161 CML161

2. CML161 G25QC18H520

3. HKI193-1 CML193

*The source of all is CCSHAU, except for CML161 which was obtained from CIMMYT, Mexico

16

Table 2: Percent polymorphism and distribution of SSRs used for background selection in the study

Chromosome

HKI323 × HKI161 HKI1105 × CML161 HKI1128 × HKI193-1

No. of markers

screened

Polymorphic

markers

No. of markers

screened

Polymorphic

markers

No. of markers

screened

Polymorphic

markers

1 43 16 (11) 43 18 (8) 44 14 (11)

2 37 15 (8) 37 18 (9) 49 20 (13)

3 40 24 (12) 40 24 (13) 46 29 (12)

4 36 12 (8) 36 15 (8) 49 19 (8)

5 32 20 (7) 32 11 (4) 33 16 (10)

6 27 19 (7) 27 11 (7) 38 22 (10)

7 42 14 (6) 42 19 (6) 40 21 (5)

8 30 9 (4) 30 8 (6) 45 13 (8)

9 38 9 (7) 38 20 (9) 50 25 (10)

10 26 8 (4) 26 10 (3) 39 7 (5)

Total 463 140 (74) 351 144 (73) 433 186 (92)

Percentage (%) - 30.2 - 41.0 - 42.9

*Figures in parenthesis indicates the number of polymorphic markers used in background selection

17

Table 3: Segregation of opaque2 allele in each of the backcross- and selfed-progenies across the three crosses

S.

No.

Cross Generation No. of plants

genotyped

SSR used

for

genotyping

Genotypic class χ2 P value

O2/O2 O2/o2 o2/o2

1.

HKI323 × HKI161

BC1F1 154

umc1066

133 21 - 81.46 <0.0001

BC2F1 152 99 53 - 13.92 0.0002

BC2F2 153 41 54 58 17.01 0.0002

2. HKI1105 × CMLI61

BC1F1 153

phi057

121 32 - 51.77 <0.0001

BC2F1 161 104 57 - 13.72 0.0002

BC2F2 155 69 66 20 34.40 <0.0001

3. HKI1128 × 1HKI193-1

BC1F1 130

umc1066

80 50 - 6.92 <0.0085

BC2F1 154 86 68 - 2.10 0.1469

BC2F2 220 91 69 60 39.30 <0.0001

18

Table 4: Recurrent parent genome (RPG) recovery and degree of opaquenessin different backcross generations

Cross Generation Genotype advanced % RPG

Recovery

Range of RPG % in selected

foreground positive plants

Opaqueness

(%)

HKI323

×

HKI161

BC1F1 HKI323-31 73.6

63.3 - 74.3 -

HKI323-44 74.3 -

BC2F1 HKI323-31-6 93.2

84.9 - 93.9 -

HKI323-44-68 93.9 -

BC2F4

HKI323-44-68-16 98.0*

91.9 - 98.7

25%

HKI323-44-68-1 94.6 25%-75%

HKI323-44-68-7 96.0 25%-75%

HKI1105

×

CMLI61

BC1F1 HKI1105-22 76.3 65.5 - 77.0

-

HKI1105-24 77.0 -

BC2F1

HKI1105-22-14 82.1 82.1 - 93.8

-

HKI1105-22-99 91.0 -

HKI1105-24-89 91.1 -

BC2F4

HKI1105-22-14-15 92.0 82.6 - 96.6

25-50%

HKI1105-22-99-3 96.6* 50%

HKI1105-24-89-31 94.3 25-50%

HKI1128

×

HKI193-1

BC1F1 HKI1128-48 90.0

80.0 - 91.9 -

HKI1128-44 85.5 -

BC2F1 HKI1128-48-1 94.0

89.6 - 94.0 -

HKI1128-44-11 93.3 -

BC2F4

HKI1128-48-1-3 96.2

91.9 - 98.4

75%

HKI1128-48-1-14 95.1* 50%

HKI1128-48-1-16 98.4 75%

* used for generating hybrid combination contributed to national testing

19

Table 5: Performance of original and improved inbreds for lysine and tryptophan in endosperm

*HKI323-Q: HKI323-44-68-16, #HKI1105-Q: HKI1105-22-99-3, $HKI1128-Q: HKI1128-48-1-14

S.

No.

Trait Year HKI323

(RP)

HKI161

(DP)

HKI323Q %

increase

over

HKI323

HKI1105

(RP)

CML161

(DP)

HKI1105Q %

increase

over

HKI1105

HKI1128 HKI193-

1 (DP)

HKI1128Q %

increase

over

HKI1128

1. % lysine in

sample

2013 0.170 0.336 0.277 63 0.195 0.373 0.341 75 0.194 0.321 0.317 63

2014 0.181 0.311 0.304 68 0.226 0.398 0.373 65 0.223 0.330 0.344 54

2. %

tryptophan

in sample

2013 0.039 0.071 0.067 72 0.052 0.079 0.076 46 0.044 0.077 0.080 82

2014 0.035 0.073 0.067 91 0.049 0.082 0.079 46 0.039 0.080 0.082 110

3. Protein %

2013 8.40 9.70 9.00 - 9.90 10.20 9.80 - 12.00 9.3 11.70 -

2014 8.50 9.80 8.80 - 9.70 10.10 9.70 - 11.70 9.2 11.40 -

4. % lysine in

protein

2013 2.02 3.46 3.07 52 1.97 3.66 3.48 95 1.62 3.45 2.71 67

2014 2.13 3.17 3.45 62 2.33 3.94 3.85 69 1.90 3.59 3.02 59

5. %

tryptophan

in protein

2013 0.46 0.73 0.74 61 0.53 0.77 0.78 47 0.37 0.83 0.68 84

2014 0.41 0.75 0.76 86 0.51 0.81 0.81 59 0.33 0.87 0.72 118

20

Table S1: Morphological characteristics of MAS-derived inbreds vis-à-vis original inbreds

S. No. Characteristics HKI323 HKI323-Q HKI1105 HKI1105-Q HKI1128 HKI1128-Q

1. Leaf: angle between blade and

stem (on leaf just above upper

ear)

Small Small Wide Wide Small Small

2. Leaf: attitude of blade Drooping Drooping Straight Straight Straight Straight

3. Stem: anthocyanin colouration

of brace root

Present Present Present Absent Present Present

4. Tassel: time of anthesis Medium Medium Medium Medium Late Late

5. Tassel: anthocyanin colouration

at base of glume

Absent Present Present Present Absent Absent

6. Tassel: anthocyanin colouration

of glumes excluding base

Present Present Present Absent Absent Absent

7. Tassel: anthocyanin colouration

of anthers

Present Present Present Absent Absent Absent

8. Tassel: density of spikelets Sparse Sparse Sparse Sparse Sparse Sparse

9. Tassel: angle between main axis

and lateral branches

Narrow Narrow Wide Wide Narrow Narrow

10. Tassel: attitude of lateral

branches

Curved Curved Straight Straight Straight Straight

11. Ear: time of silk emergence Medium Medium Medium Medium Late Late

12. Ear: anthocyanin colouration of

silks

Present Present Absent Present Absent Absent

13. Leaf: anthocyanin colouration of

sheath below ear

Absent Absent Absent Absent Absent Absent

14. Tassel: length of main axis

above lowest side branch

Long Long Medium Medium Long Long

15. Plant length up to flag leaf Medium Medium Short Short Long Long

16. Plant: ear placement Medium Medium Medium Medium Medium Medium

21

S. No. Characteristics HKI323 HKI323-Q HKI1105 HKI1105-Q HKI1128 HKI1128-Q

17. Leaf: width of blade Medium Medium Broad Broad Medium Medium

18. Ear: length without husk Medium Medium Medium Medium Long Long

19. Ear: diameter Small Small Small Small Small Small

20. Ear: shape Conical Conical Cylindrical Cylindrical Conical Conical

21. Ear: number of rows of grains Many Many Medium Medium Many Many

22. Ear: type of grain Flint Flint Flint Flint Flint Flint

23. Ear: colour of top of grain Orange Orange Orange Yellow with cap Orange Yellow with cap

24. Ear: colouration of glumes of

cob

White White White White White White

25. Kernel: row arrangement Straight Straight Straight Straight Straight Straight

26. Kernal: poppiness Absent Absent Absent Absent Absent Absent

27. Kernal: sweetness Absent Absent Absent Absent Absent Absent

28. Kernal: waxiness Absent Absent Absent Absent Absent Absent

29. Kernel: opaqueness Absent Present Absent Present Absent Present

30. Kernel: shape Round Round Round Round Round Round

31. Kernel: 1000 kernel weight Medium Medium Medium Medium Medium Medium

*HKI323-Q: HKI323-44-68-16, #HKI1105-Q: HKI1105-22-99-3, $HKI1128-Q: HKI1128-48-1-14

22

Table 6: Agronomic performance of improved inbreds vis-à-vis original inbreds in Delhi during rainy season-2013 and -2014

S.

No.

Genotype Year of

testing

MF

(d)

FF

(d)

PH

(cm)

EH

(cm)

TL

(cm)

LBW

(cm)

CL

(cm)

CG

(cm)

NR NKR 100KW

(g)

YLD

(kg/ha)

1. HKI323

2013 49.0 51.0 137.5 72.2 30.0 7.1 12.7 3.2 11.7 19.2 24.8 2370

2014 48.0 50.0 139.2 71.7 36.0 8.0 12.3 3.3 12.0 20.0 26.5 3015

2. HKI323Q

2013 51.5 53.5 130.0 64.2 32.2 8.4 13.3 3.5 13.3 18.7 24.4 2335

2014 48.5 50.0 140.0 69.2 34.8 8.0 12.4 3.3 12.0 18.8 26.1 3389

3. HKI1105

2013 52.5 52.5 107.5 65.7 25.4 10.1 14.3 3.5 13.0 20.8 26.6 2096

2014 51.0 51.5 114.2 75.0 31.3 10.2 14.3 3.4 13.0 21.0 25.2 2765

4. HKI1105Q

2013 49.5 51.0 100.0 50.8 25.4 9.8 13.8 3.3 12.0 19.8 24.5 1920

2014 51.0 51.5 111.7 66.7 28.3 9.9 14.1 3.3 13.7 20.8 24.3 2795

5. HKI1128

2013 53.5 55.5 155.8 86.3 31.5 7.0 14.4 3.3 13.0 21.3 21.3 1595

2014 51.0 51.0 162.5 88.3 36.5 7.4 14.8 3.3 12.7 22.2 23.1 3048

6. HKI1128Q

2013 51.5 53.5 165.0 84.2 33.4 7.8 15.3 3.1 12.7 21.3 22.4 1599

2014 50.5 51.0 160.0 84.2 37.5 7.8 14.9 3.3 13.0 22.0 22.4 2959

SE 2013 0.72 0.72 10.72 5.56 1.45 0.55 0.38 0.07 0.26 0.46 0.78 142.97

2014 0.57 0.28 9.03 3.59 1.48 0.49 0.48 0.02 0.27 0.53 0.68 93.63

*HKI323-Q: HKI323-44-68-16, #HKI1105-Q: HKI1105-22-99-3, $HKI1128-Q: HKI1128-48-1-14; MF: days to 50% male flowering; FF: days to 50% female

flowering; PH: plant height; EH: ear height; TL: tassel length; LBW: width of leaf blade; CL: cob length; CG: cob girth; NR: No. of rows; NKR: No. of

kernel per row; 100KW: 100 kernel weight; YLD: Grain yield; d: days

23

Table 7: Performance of original and improved hybrids for lysine and tryptophan in endosperm

S.

No.

Quality

attributes

Year HM4 HM4-Q % increase

over HM4

HM8 HM8-Q % increase

over HM8

HM9 HM9-Q % increase

over HM9

1. % lysine in

sample

2013 0.155 0.232 50 0.194 0.340 75 0.202 0.340 68

2014 0.162 0.245 51 0.191 0.321 68 0.197 0.351 78

2. % tryptophan

in sample

2013 0.032 0.064 100 0.046 0.081 76 0.045 0.068 51

2014 0.036 0.070 94 0.044 0.085 93 0.043 0.071 65

3. Protein %

2013 8.60 8.70 - 8.60 8.80 - 8.90 8.60 -

2014 8.70 8.70 - 8.80 8090 - 9.0 8.90 -

4. % lysine in

protein

2013 1.80 2.67 48 2.26 3.86 71 2.27 3.95 74

2014 1.86 2.81 51 2.17 3.61 66 2.19 3.94 80

5. % tryptophan

in protein

2013 0.37 0.74 100 0.53 0.92 74 0.51 0.79 55

2014 0.41 0.80 95 0.50 0.96 92 0.48 0.80 67

HM4-Q: HKI1105-22-99-3 × HKI323-44-68-16; HM8-Q: HKI1105-22-99-3 × HKI161; HM9-Q: HKI1105-22-99-3 × HKI1128-48-1-14

24

Table S2: Morphological characteristics of reconstituted hybrids vis-à-vis original- hybrids

S. No. Characteristics HM4 HM4-Q HM8 HM8-Q HM9 HM9-Q

1. Leaf: angle between blade and

stem (on leaf just above upper

ear)

Small Small Small Small Small Small

2. Leaf: attitude of blade Straight Straight Straight Straight Straight Straight

3. Stem: anthocyanin colouration

of brace root

Present Present Present Present Present Present

4. Tassel: time of anthesis Medium Medium Medium Medium Medium Medium

5. Tassel: anthocyanin colouration

at base of glume

Present Present Present Absent Absent Absent

6. Tassel: anthocyanin colouration

of glumes excluding base

Absent Present Present Present Absent Absent

7. Tassel: anthocyanin colouration

of anthers

Present Present Present Present Absent Present

8. Tassel: density of spikelets Sparse Sparse Sparse Sparse Sparse Sparse

9. Tassel: angle between main

axis and lateral branches

Narrow Narrow Narrow Narrow Narrow Narrow

10. Tassel: attitude of lateral

branches

Curved Curved Straight Curved Straight Straight

11. Ear: time of silk emergence Medium Medium Medium Medium Medium Medium

12. Ear: anthocyanin colouration of

silks

Present Present Present Present Absent Present

13. Leaf: anthocyanin colouration

of sheath below ear

Absent Absent Absent Absent Absent Absent

14. Tassel: length of main axis

above lowest side branch

Long Long Long Long Long Long

15. Plant length up to flag leaf Medium Medium Medium Medium Long Long

16. Plant: ear placement Medium Medium Medium Medium Medium Medium

25

S. No. Characteristics HM4 HM4-Q HM8 HM8-Q HM9 HM9-Q

17. Leaf: width of blade Broad Broad Broad Broad Medium Medium

18. Ear: length without husk Long Long Long Long Long Long

19. Ear: diameter Medium Medium Medium Medium Medium Medium

20. Ear: shape Cylindrical Cylindrical Cylindrical Cylindrical Cylindrical Cylindrical

21. Ear: number of rows of grains Many Many Many Many Many Many

22. Ear: type of grain Flint Flint Flint Flint Flint Flint

23. Ear: colour of top of grain Orange Yellow with cap Orange Yellow with cap Orange Yellow with cap

24. Ear: colouration of glumes of

cob

White White White White White White

25. Kernel: row arrangement Straight Straight Straight Straight Straight Straight

26. Kernel: poppiness Absent Absent Absent Absent Absent Absent

27. Kernel: sweetness Absent Absent Absent Absent Absent Absent

28. Kernel: waxiness Absent Absent Absent Absent Absent Absent

29. Kernel: opaqueness Absent Present Present Present Absent Present

30. Kernel: shape Round Round Round Round Round Round

31. Kernel: 1000 kernel weight Medium Medium Medium Medium Medium Medium

26

Table 8: Agronomic performance of improved hybrids vis-à-vis original hybrids at Delhi during kharif 2013

S.

No

Genotype MF

(d)

FF

(d)

PH

(cm)

EH

(cm)

TL

(cm)

LBW

(cm)

CL

(cm)

CG

(cm)

NR NKR 100KW

(g)

YLD

(kg/ha)

1. HM4 44.0 45.0 181.7 40.3 107.5 11.6 17.6 3.9 13.3 34.7 30.8 7160

2. HM4-Q 44.5 45.5 175.0 38.3 106.7 11.5 17.3 4.0 13.3 37.0 29.0 7042

3. HM8 45.0 45.0 158.3 36.7 93.3 11.0 19.1 4.6 14.0 34.8 32.2 6649

4. HM8-Q 46.0 46.5 167.5 36.2 93.3 10.9 18.9 4.6 14.3 37.5 30.3 6954

5. HM9 44.5 45.5 171.7 38.6 101.7 9.1 20.5 4.3 14.3 36.0 28.2 6347

6. HM9-Q 44.5 45.5 178.3 42.1 105.8 8.3 21.2 4.4 13.3 40.7 28.0 6084

SE 0.29 0.23 3.49 0.92 2.74 0.57 0.64 0.12 0.21 0.93 0.68 176.73

MF: days to 50% male flowering; FF: days to 50% female flowering; PH: plant height; EH: ear height; TL: tassel length; LBW: width of leaf

blade; CL: cob length; CG: cob girth; NR: No. of rows; NKR: No. of kernel per row; 100KW: 100 kernel weight; YLD: Grain yield; d: days

27

Table S3: Mean phenotypic performance of reconstituted hybrids vis-à-vis original hybrids at multi-locations under AICRP trials during 2014- and

2015- rainy season

S.

No.

Traits Years HM4 HM4-Q CD HM8 HM8-Q CD HM9 HM9-Q CD

1. Plant height (cm) 2014 182.8 (9) 178 (9) 11.09 194.7 (12) 187.7 (12) 8.5 170.1 (7) 172.1 (7) 6.23

2015 178.8 (4) 170.4 (4) 20.66 185.0 (5) 203.0 (5) 11.0 177.7 (5) 179.0 (5) 16.74

Weighted mean 181.6 175.7 - 191.9 192.2 - 173.3 175.0 -

2. Days to 50%

pollen shedding

2014 50.8 (9) 49.3 (9) 1.77 56.1 (12) 56.9 (12) 1.12 52.3 (7) 53.5 (7) 1.08

2015 52.9 (4) 52.3 (4) 2.73 52.7 (5) 54.9 (5) 2.19 50.7 (5) 52.5 (5) 2.23

Weighted mean 51.5 50.2 - 55.1 56.3 - 51.6 53.1 -

3. Days to 50%

silking

2014 53.8 (9) 52.2 (9) 1.75 57.8 (12) 59.2 (12) 1.18 55.2 (7) 56.9 (7) 1.33

2015 56.0 (4) 54.1 (4) 3.10 54.6 (5) 56.5 (5) 2.34 54.5 (5) 55.5 (5) 3.07

Weighted mean 54.5 52.8 - 56.9 58.4 - 54.9 56.3 -

4. Days to 75% dry

husk

2014 88.8 (9) 86.8 (9) 2.40 95.1 (10) 96.3 (10) 2.20 83.8 (7) 90.3 (7) 6.84

2015 91.4 (3) 86.9 (3) 6.38 92.1 (4) 93.2 (4) 3.33 87.8 (5) 86.7 (5) 3.01

Weighted mean 89.5 86.8 - 94.2 95.4 - 85.5 88.8 -

5. Grain yield (kg/ha) 2014 5257 (9) 6233 (9) 997 5775 (12) 6194 (12) 1306 4819 (7) 5081 (7) 733

2015 6951 (4) 6836 (4) 782 6356 (5) 6412 (5) 885 4913 (5) 5369 (5) 884

Weighted mean 5778 6419 - 5946 6258 - 4858 5201

Values in parenthesis indicate number of locations the hybrids was tested

28

Table S4: Responses of reconstituted hybrids vis-à-vis original hybrids to various biotic stresses at multi-locations under AICRP trials during 2014-

and 2015- rainy season

Disease Year No. of locations HM4 HM4-Q HM8 HM8-Q HM9 HM9-Q

MLB 2014 5 3.0 (MR) 2.4 (MR) 3.0 (MR) 2.7 (MR) 2.8 (MR) 2.3 (MR)

2015 4 2.7 (MR) 2.1 (R) 1.9 (R) 2.4 (MR) 2.0 (R) 1.8 (R)

TLB 2014 4 2.9 (MR) 2.3 (MR) 4.4 (S) 2.5 (MR) 3.0 (MR) 2.9(R)

2015 5 2.8 (MR) 2.3 (MR) 2.3 (MR) 2.4 (MR) 2.5 (MR) 2.3 (MR)

BLSB 2014 6 3.3 (MS) 3.8 (MS) 3.1 (MS) 3.7 (MS) 3.3 (MS) 3.5 (MS)

2015 4 3.7 (MS) 3.7 (MS) 4.0 (MS) 3.8 (MS) 3.9 (MS) 3.5 (MS)

Polysora rust 2014 1 2.5 (MS) 2.8 (MS) 3.5 (S) 3.3 (S) 2.3 (MS) 2.8 (MS)

2015 1 2.8 (MS) 3.3 (S) 2.3 (MS) 2.3 (MS) 2.5 (MS) 2.8 (MS)

Common rust 2014 1 3.5 (S) 3.0 (MS) 5.0 (HS) 3.0 (MS) 2.5 (MS) 4.0 (S)

2015 1 3.5 (MS) 4.0 (S) 1.0 (R) 1.0 (R) 2.0 (MR) 2.0 (MR)

Charcoal rot 2014 2 4.8 (MR) 3.7 (MR) 6.0 (MS) 5.1 (MS) 3.3 (MR) 3.4 (MR)

2015 3 5.0 (MR) 5.8 (MS) 4.2 (MR) 5.2 (MS) 5.2 (MS) 5.5 (MS)

FSR 2014 1 3.4 (MR) 1.8 (R) 2.5 (R) 1.2 (R) 2.8 (R) 2.4 (R)

2015 1 3.7 (MR) 2.6 (MR) 2.9 (R) 3.2 (MR) 4.0 (MR) 2.7 (R)

SDM 2014 1 100 (S) 100 (S) 100 (S) 100 (S) 100 (S) 100 (S)

2015 1 100 (S) 100 (S) 100 (S) 100 (S) 100 (S) 100 (S)

RDM 2014 1 0.0 (R) 81.0 (S) 29.0 (MS) 81 (S) 12.0 (MR) 50.0 (MS)

2015 1 15.0 (MR) 13.0 (MR) 1.5 (R) 2.0 (R) 6.0 (R) 11.0 (MR)

BSR 2014 2 31.4 (MS) 45.6 (MS) 39.3 (MS) 37.5 (MS) 26.1 (MS) 48.0 (MS)

2015 2 21.7 (MR) 23.5 (MR) 17.1 (MR) 25.1 (MS) 14.6 (MR) 11.7 (MR)

MLB: maydis leaf blight; TLB: turcicum leaf blight; BLSB: banded leaf and sheath blight; FSR: Fusarium stalk rot; SDM: sorghum downy mildew; RDM:

Rajasthan downy mildew; BSR: bacterial stalk rot; numerical value represents average disease score over locations; value in parenthesis represents type of

reactions. R: resistant, MR: moderately resistant, MS: moderately susceptible; S: susceptible; HS: highly susceptible